Methods and systems for starch based propagation of a microorganism

ABSTRACT

Systems and methods that include starch derived from grain as a carbon source for propagation of microorganisms.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/310,173 filed Dec. 14, 2018, Published as US-2019-0256873-A1 on Aug.22, 2019, which is a 371 Application of PCT/US2017/036958 filed Jun. 12,2017, which claims the benefit of U.S Provisional Patent Application No.62/351,036, filed on Jun. 16, 2016, wherein the entirety of each of saidpatent document is incorporated herein by reference.

FIELD

The present invention relates to using starch based propagation media toreproduce microorganisms such as yeast cells used in cellulosic ethanolproduction.

SUMMARY

The present disclosure includes embodiments of a method of producing abiochemical, the method including:

a) providing a propagation composition including one or more saccharidesderived from grain, and a first cell mass of a microorganism;

b) exposing the propagation composition to conditions to propagate thefirst cell mass of the microorganism into a second cell mass of themicroorganism greater than the first cell mass of the microorganism;

c) providing a fermentation composition including one or moresaccharides derived from lignocellulosic feedstock and one or moretoxins that are toxic to the microorganism, and at least a portion ofthe second cell mass of microorganism; and

d) exposing the fermentation composition to conditions so that the atleast a portion of the second cell mass of microorganism convertssaccharides in the fermentation composition into a biochemical.

The present disclosure also includes embodiments of a method ofpropagating a microorganism, the method including:

a) providing a propagation composition including one or morepolysaccharides, a first cell mass of a microorganism that can convertmonosaccharide into a biochemical, and one or more enzymes that canconvert polysaccharide into one or more monosaccharides;

b) exposing the propagation composition to conditions to convertpolysaccharide into one or more monosaccharides, and propagate the firstcell mass of the microorganism into a second cell mass of themicroorganism;

c) providing a lignocellulosic hydrolysate including one or moremonosaccharides and one or more toxins that are toxic to themicroorganism; and d) combining the lignocellulosic hydrolysate and atleast a portion of the second cell mass of microorganism so that thesecond cell mass of micororganism can convert monosaccharide into abiochemical.

The present disclosure also includes embodiments of a biorefineryincluding:

a) a propagation system including at least one vessel that contains apropagation composition, wherein the propagation composition includesone or more saccharides derived from grain and a first cell mass of amicroorganism, wherein the propagation system is configured to exposethe propagation composition to conditions to propagate the first cellmass of the microorganism into a second cell mass of the microorganismgreater than the first cell mass of the microorganism; and

b) a fermentation system, wherein the fermentation system is in fluidcommunication with the propagation system to receive at least a portionof the second cell mass of the microorganism, wherein the fermentationsystem is also in fluid communication with a source of a fermentationcomposition including saccharides derived from lignocellulosic feedstockand one or more toxins that are toxic to the microorganism, and whereinthe fermentation system is configured to combine the fermentationcomposition and the at least a portion of the second cell mass of themicroorganism so that the second cell mass of the microorganism convertssaccharide in the fermentation composition into a biochemical.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a process flow diagram for propagating a microorganism thatcan be used in a fermentation system to convert one or moremonosaccharides into one or more biochemicals.

FIG. 2 shows a process flow diagram of an embodiment according to thepresent disclosure.

FIG. 3A shows a graph for data in Example 1;

FIG. 3B shows another graph for data in Example 1;

FIG. 3C shows another graph for data in Example 1;

FIG. 4A shows another graph for data in Example 1;

FIG. 4B shows another graph for data in Example 1;

FIG. 4C shows another graph for data in Example 1;

FIG. 5A shows another graph for data in Example 1;

FIG. 5B shows another graph for data in Example 1;

FIG. 5C shows another graph for data in Example 1;

FIG. 6 shows a graph for data in Example 2;

FIG. 7A shows a graph for data in Example 3;

FIG. 7B shows another graph for data in Example 3; and

FIG. 7C shows another graph for data in Example 3.

DETAILED DESCRIPTION

Disclosed in embodiments herein are methods and systems for using grainstarch (e.g., corn grain starch) to propagate a “seed” microorganisminto a larger population of microorganisms. Propagating a “seed”microorganism can also be referred to as “seed fermentation.” Afterpropagation (“seed fermentation”), the larger population ofmicroorganisms can be added into cellulosic hydrolysate (e.g.,hydrolysate derived from corn cobs and corn stover) so as to convert oneor more monosaccharides into one or more biochemicals. Advantageously,methods and systems according to the present disclosure can help createbetter and healthier yeast during propagation that can then be used incellulosic ethanol systems.

As shown in FIG. 1, a system for propagating a microorganism can includecombining at least a carbon source, a nutrient source, and a first cellmass of a microorganism under conditions to reproduce in number and forma second cell mass that is greater in cell number as compared to thefirst cell mass. Optionally, one or more additional components can beadded to the propagation system such as water and the like.Microorganisms

Microorganisms that can convert one or more monosaccharides into abiochemical include, e.g., bacteria and/or fungi such as yeast. Theproduct biochemicals can vary depending on the conditions that areprovided. In many embodiments, the biochemicals include biofuels such asethanol, butanol, and the like. In some embodiments, the microorganismincludes one or more ethanologenic microorganisms referred to as“ethanologens”. As used herein, an “ethanologen” refers to amicroorganism that can convert one or more monosaccharides (e.g.,glucose and the like) into at least ethanol.

Exemplary yeast and fungus include the genus of, Aspergillus, Candida,Pichia, (Hansenula), Phanerochaete, Kloeckera (Hanseniaspora),Kluyveromyces, Rhodotorula, Torulopsis, Zygosaccharomyces, Yarrowia, andSaccharomyces. In some embodiments, the yeast is a strain ofSaccharomyces cerevisiae yeast. In some embodiments, the microorganismto be propagated includes genetically modified yeast such as geneticallymodified Saccharomyces cerevisiae that can convert glucose and xyloseinto a biochemical such as ethanol.

Nutrient Source

A nutrient source is also included to help propagate microorganisms suchas ethanologens. As used herein, a “nutrient source” refers to one ormore materials that can be used by a microorganism to grow and/orreproduce to create additional microorganisms and is different from acarbon source or can be used as a carbon source as well.

In some embodiments, the nutrient source used includes a stillagecomposition (e.g. whole stillage, thin stillage and/or syrup). Wholestillage is a byproduct of distilling a fermentation product. Forexample, a process for making whole stillage is a corn grain-to-ethanolprocess. For example, a grain such as corn, barley, wheat, and/orsorghum can be fermented into a fermentation product that includes oneor more biochemicals such as ethanol. Either the ground whole grain canbe used or only one or more portions of the grain can be used. Forexample, whole grains can be dry milled for fermentation or fractionatedinto one or more separated portions before milling. After milling, themilled grain material can be further processed to break downpolysaccharides and/or oligosaccharides into one or more monosaccharidessuch as glucose that can be fermented by, e.g., yeast. Methods ofbreaking down polysaccharides such as starch into glucose include e.g.hot water, such as hot water that includes an added acid such assulfuric acid, and/or enzymatic pretreatment. After fermentation, thefermentation product is distilled in a system where the ethanol isremoved from the fermented mash in a distillation column. After theethanol is removed, the remaining residue is removed as stillageresidue. The stillage residue is known as “whole stillage.” The wholestillage can be optionally further processed via one or more systems tofurther clarify or separate the whole stillage before being delivered toa propagation system such as in FIG. 1. For example, the whole stillagecan be subjected to a solid-liquid separation process to produce a solidstream of residue, also known as wet cake, and a liquid stream ofresidue, also referred to as thin stillage. The thin stillage can befurther processed to increase the solids concentration by evaporationresulting in condensed distillers solubles or syrup.

Such stillage composition from the grain-to-ethanol producing process,including the whole stillage, wet cake, thin stillage, and/or syrup canbe used as at least part of the nutrient source for propagatingmicroorganisms such as yeast. Using at least a portion of the wholestillage provides an alternative or additional nutrient source ascompared to, e.g., yeast extract. Using whole stillage (e.g. thinstillage) as the entire amount of nutrients or part of the nutrients canpropagate yeast as well as, or better than, other nutrient sources suchas yeast extract.

In some embodiments, the nutrient source includes a stillage compositionsuch as thin stillage, wetcake, syrup, and any combination thereof. Thenutrient source can include syrup derived from thin stillage, thinstillage, or combinations thereof.

The stillage composition can be provided in any amount so as to helpreproduce (propagate) and generate a desired population of microorganism(e.g., ethanologen) within a given amount of time. The amount ofstillage component provided can depend on factors such as the type andamount of other nutrient sources present, the type and amount of carbonsources present, pH, temperature, desired time period for propagation,and the like. In some embodiments, the nutrient source includes onlythin stillage.

In some embodiments, the thin stillage can be provided to thepropagation system at a temperature in the range from 70 to 95° C., oreven from 80 to 90° C.

Carbon Source

As used herein, a “carbon source” refers to one or more compounds thatinclude at least one carbon atom and can be used by a microorganism suchas yeast to grow and/or reproduce to create additional biomass.Exemplary carbon sources include monosaccharides such as glucose,fructose, galactose, mannose, xylose and the like; disaccharides such aslactose, maltose, sucrose, cellobiose and the like; oligosaccharides;polysaccharides such as cellulose, hemicelluloses, starch, xylane andthe like; single carbon substrates including only one carbon atom suchas methanol; and polyols such as glycerol, but not limited thereto.

In a cellulosic process that uses biomass such as corn cobs and/or cornstover, the carbon source can include a hydrolysate from acidhydrolysis, steam explosion, enzymatic liquefaction, and/or enzymaticsaccharification. Unfortunately such sources of a carbon source caninclude components that are toxic (toxins) to microorganisms such asyeast. Such toxins include furfural, hydroxymethylfurfural, acetic acid,and the like. Using yeast and furfural as an example for illustrationpurposes, yeast tend to degrade at least a portion of furfural beforeperforming other metabolic activity such as propagation and/orfermentation. Thus, especially in a large scale commercial operation,yeast may spend an undue amount of time (e.g., 24 hours) degradingfurfural before propagating yeast into additional yeast cells. This canmake propagation economically undesirable and/or put a strain on theyeast and may cause a less healthy yeast population to be produced.Further, bacterial contamination (e.g., lactic acid bacteria) is oftenpresent in propagation, fermentation, and the like. Such bacteria cancompete with the yeast for sugar and can have a “head start” relative tothe yeast in consuming sugar while the yeast is degrading furfural. Sucha competition for sugar can be an additional strain on the yeast and maycause a less healthy yeast population to be produced.

Accordingly, there is a continuing desire to identify a propagationmedia substantially free of furfural for yeast propagation and/or tobetter out-compete bacterial contamination, especially in the context ofcellulosic fermentation process. Also desired are methods and system togenerate better quality (“healthier”) and/or greater yeast quantity tomore quickly detoxify (degrade) furfural that is eventually encounteredby the yeast during fermentation of a cellulosic hydrolysate (e.g., cornstover and corn cob hydrolysate) to speed up fermentation, improvefermentation, and/or out-compete bacterial contamination.

An alternative propagation media has been discovered where at least thecellulosic saccharification broth used as carbon source in thepropagation composition (medium) is replaced at least in part by starchethanol plant corn slurry. Starch ethanol plant corn slurry issubstantially free of furfural. Traditional alpha-amylases andgluco-amylases that are commercially available may be used hydrolyse thestarch to form the glucose for propagation.

Embodiments of the present disclosure include a carbon source such asstarch derived from a grain such as corn. The starch can be hydrolyzedinto glucose prior to being combined with yeast for propagation orin-situ as the yeast is being propagated. Further, at least a portion ofthe starch can be hydrolyzed prior to adding yeast while the remainderof the starch is hydrolyzed in the presence of yeast. Hydrolyzing grainstarch such as corn starch can be performed in a manner that avoidsgeneration of toxins such as furfural. Advantageously, the yeast canavoid having to degrade furfural, thereby avoiding lag time associatedwith degrading the furfural. Thus, the head start on sugar consumptionby bacteria (discussed above) can be reduced or avoided completely. Inaddition, while not being bound by theory, it is believed that the yeastare not unduly stressed during propagation due to having to degradefurfural and, thus, a relatively healthier, more robust, yeastpopulation is created that can better withstand eventual exposure totoxins such as furfural that may be present in hydrolysate compositionssuch as those derived from corn stover and corn cobs. Propagation

FIG. 2 illustrates an embodiment according to the present disclosure.FIG. 2 represents a biorefinery having a corn-grain ethanol processco-located with a cellulosic ethanol process. Such processes can bephysically located relatively near each other such as within severalhundred yards such that piping and like can be easily installed betweenthe two processes. As shown in FIG. 2, ground grain slurry 126 isprovided to a propagation system 140 from a corn starch ethanol process131. The ground grain slurry can include ground corn grain slurry thatincludes corn starch. In some embodiments, the ground corn grain isformed by dry grinding whole corn grain in corn ethanol process 131. Insome embodiments, the whole ground corn grain can be combined with waterand thin stillage to form a pumpable slurry that can be pumped topropagation system 140. In some embodiments, one or more pipe lines usedto pump the corn slurry can be flushed with water such as distillatefrom corn ethanol process 131. Such water can be used to make apropagation composition in the propagation system 140. Propagationsystem 140 can include at least one vessel in fluid communication withthe ground grain slurry 126 so that the grain slurry can be added to theat least one vessel. In some embodiments, ground grain slurry 126, anutrient source (e.g. thin stillage), a first cell mass of amicroorganism (e.g., yeast), and at least one alpha-amylase can be addedto the at least one vessel of the propagation system 140 and held at atemperature in the range from 65° C. to 80° C. (e.g., about 70° C.) fora time period in the range from 1 to 3 hours. During this time period,the alpha-amylase can facilitate converting at least a portion of thestarch to one or more oligosaccharides. Also, this temperature holdingperiod can help pasteurize at least a portion of any bacterialcontamination that may be present. Advantageously, includingpasteurization and avoiding furfural in this manner can help the yeastpropagate sooner and with less competition for glucose.

In some embodiments, the contents of the at least one vessel can becooled to a temperature in the range from 20° C. to 40° C. (e.g., about31° C.). During and/or after cooling, gluco-amylase can be added tofacilitate converting one or more oligosaccharides into glucose so thatthe first cell mass of the microorganism can utilize the glucose topropagate into the second cell mass of the microorganism. In someembodiments, a propagation vessel can be coupled to an aerator tofacilitate propagation. A propagation composition can be held at atemperature in the range from 20° C. to 40° C. (e.g., about 31° C.) fora time period of from 10-20 hours (e.g., about 15-16 hours).

In some embodiments, the second cell mass of the microorganism is atleast 500 times greater in number than the first cell mass of themicroorganism, at least 1000 times greater in number than the first cellmass of the microorganism, at least 1500 times greater in number thanthe first cell mass of the microorganism, at least 2000 times greater innumber than the first cell mass of the microorganism, or even at least3000 times greater in number than the first cell mass of themicroorganism.

In some embodiments, the first cell mass of the microorganism is5×10{hacek over ( )}⁶ cells per milliliter of propagation composition orless, 1×10{hacek over ( )}⁶ cells per milliliter of propagationcomposition or less, 5×10{hacek over ( )}⁵ cells per milliliter ofpropagation composition or less, or even 1×10{hacek over ( )}⁵ cells permilliliter of propagation composition or less.

In some embodiments, the second cell mass of the microorganism is1×10{hacek over ( )}⁷ cells per milliliter of propagation composition ormore, 5×10{hacek over ( )}⁷ cells per milliliter of propagationcomposition or more, 1×10{hacek over ( )}⁸ cells per milliliter ofpropagation composition or more, 5×10{hacek over ( )}⁸ cells permilliliter of propagation composition or more, or even 1×10{hacek over( )}⁹ cells per milliliter of propagation composition or more.

Optionally, one or more additional components can be added to the atleast one vessel to form a propagation composition. For example, processwater from the cellulosic ethanol process and/or hot distillate from acorn ethanol process and/or cellulosic ethanol process can be added ifmore water is desired.

In some embodiments, a propagation composition in propagation system 140can include between 5 to 25% by volume of ground corn slurry; between20-30% by volume of thin stillage as a nutrient source; enzymes; yeast;and the balance water (e.g., cellulosic process water and/or cornethanol distillate).

After propagation in propagation system 140, the propagation compositionincluding the second cell mass of yeast 141 can be combined with alignocellulosic hydrolysate in whole broth fermentation system 150 sothat the second cell mass of organism can convert glucose and/or xyloseinto a biochemical in a continuous manner.

The remainder of the cellulosic ethanol system 100 in FIG. 2 isdiscussed below. Before hydrolysis, a lignocellulosic feedstock can beprepared by a variety of techniques such as size reduction, steaming,combinations of these, and the like. As shown in FIG. 2, lignocellulosicfeedstock 105 can be prepared prior to hydrolysis such as by grindingthe lignocellulosic feedstock in one or more grinders 110 into groundsolids 115 to reduce the size of the feedstock and increase its surfacearea for contact with a hydrolysis medium.

Acid Hydrolysis

FIG. 2 shows an embodiment of hydrolyzing lignocellulosic feedstock bypassing a whole broth hydrolysate from acid hydrolysis 120 to enzymaticsaccharification 135.

As shown in FIG. 2, acid hydrolysis 120 can convert hemicellulose in theground biomass 115 into one or more pentoses such as xylose. In someembodiments, the acid hydrolysis includes contacting lignocellulosicbiomass with an aqueous composition to hydrolyze at least a portion ofthe hemicellulose into one or more oligosaccharides and/or one or morepentoses, and form a first whole broth hydrolysate composition includingat least pentose, cellulose, lignin, and furfural. In some embodiments,acid hydrolysis hydrolyzes at least a portion of cellulose into glucose.

During acid hydrolysis, the “severity” can be adjusted by varying one ormore of time period, temperature, and pH of hydrolysis. In someembodiments, during hydrolysis an aqueous composition can have a pH inthe range from 1 to 5, or even 2 to 3. The aqueous composition caninclude an acid such as sulfuric acid present in a concentration in therange from 0.2 to 1.3% w/w, or even 0.5 to 1% w/w. In some embodiments,acid hydrolysis can be performed for a time period in a range from 15minutes to 5 hours, or even 30 minutes to 4 hours. In some embodiments,acid hydrolysis can be performed at a temperature in the range fromgreater than 100° C. to 170° C., or even from 110° C. to 150° C.

Acid hydrolysis can be performed in a variety of system and apparatusconfigurations. In some embodiments, an acid hydrolysis system caninclude a first reactor system in fluid communication with a source oflignocellulosic biomass and a source of an aqueous composition. Thefirst reactor system can include at least one reactor configured tocontact the lignocellulosic biomass with the aqueous composition tohydrolyze at least a portion of the hemicellulose into one or moreoligosaccharides and/or one or more pentoses, and form a first, wholebroth hydrolysate composition including at least pentose, cellulose,lignin, and furfural.

Optional Steam Explosion

Optionally, the whole broth hydrolysate 125 from acid hydrolysis 120 canbe subjected to steam explosion conditions that make the cellulose inthe whole broth hydrolysate 125 more accessible during enzymatichydrolysis 135. In some embodiments, steam explosion also formsfurfural. Steam explosion (not shown) can be performed in a system thatincludes at least one reactor configured to receive the hydrolysatecomposition and subject the cellulose in the hydrolysate composition toa steam explosion process under conditions that form a steam-exploded,hydrolysate composition including at least cellulose, lignin, andfurfural.

During steam explosion, cellulose (either in a whole broth hydrolysateor hydrolysate with a portion of xylose liquor removed) can be subjectedto a relatively elevated pressure and temperature so that moistureimpregnated within the cellulose network is in a liquid state. Then, thepressure can be reduced so that the liquid “flashes” to a gas state sothat the sudden expansion with the cellulose network causes at least aportion of the cellulose structure to rupture, thereby increasing thesurface area of the cellulose for increased exposure to cellulaseenzymes. In some embodiments, the superheated hydrolysate compositioncan be flashed to a reduced pressure by continuously discharging thecomposition through an orifice. In some embodiments, a hydrolysatecomposition including cellulose can be subjected to a temperature in therange from 320° F. to 400° F. and a pressure in the range from 75 psigto 235 psig, followed by suddenly exposing the hydrolysate compositionto a reduced pressure such as atmospheric pressure. In some embodiments,a hydrolysate composition including cellulose can be subjected to atemperature in the range from 350° F. to 385° F. and a pressure in therange from 120 psig to 195 psig, followed by suddenly exposing thehydrolysate composition to a reduced pressure such as atmosphericpressure.

After steam explosion, the solid cellulose in the hydrolysate can besubjected to enzymatic hydrolysis 135.

Enzymatic Hydrolysis

As shown in FIG. 2, after acid hydrolysis 120 and optional steamexplosion, at least a portion of the cellulose in the hydrolysatecomposition can be enzymatically hydrolyzed 135 to hydrolyze thecellulose in into glucose. In some embodiments, as shown in FIG. 2, atleast a portion of the cellulose in the first whole broth hydrolysatecomposition 125 provided directly from acid hydrolysis 120 can beenzymatically hydrolyzed. In some embodiments, enzymatic hydrolysis 135can include liquefying (liquefaction) at least a portion of thecellulose in the hydrolysate 125 followed by saccharifying(saccharification) at least a portion of the liquefied cellulose to formglucose. Liquefaction can include adding one or more cellulase enzymesto the whole broth hydrolysate composition 125 to liquefy at least aportion of the cellulose. A liquefaction system can include one or morevessels (not shown) containing a whole broth hydrolysate and configuredto maintain the whole broth hydrolysate at a pH and temperature for atime period to convert at least a portion of the cellulose in thelignocellulosic biomass into an oligosaccharide and/or a monosaccharide.In some embodiments, the temperature of the whole broth hydrolysateduring at least a portion of liquefaction is in a range from 45° C. to65° C., or even from 50° C. to 60° C. In some embodiments, the pH of thewhole broth hydrolysate during at least a portion of liquefaction is ina range from 4 to 6, or even from 4.5 to 5.5. In some embodiments, theliquefaction time period is in the range from 2 to 20 hours, or evenfrom 6 to 8 hours.

A saccharification system can be in fluid communication with theliquefaction system. In some embodiments, a saccharification system caninclude at least one reactor configured to receive the liquefiedcellulose so as to saccharify at least a portion of the liquefiedcellulose and form glucose. A saccharification system can include one ormore batch reactors (not shown) in fluid communication with theliquefaction system to receive the liquefied cellulose. Thesaccharification system can be configured to maintain a whole brothhydrolysate at a pH and a temperature for a time period to convert atleast a portion of the cellulose in the lignocellulosic biomass into anoligosaccharide and/or a monosaccharide. In some embodiments, thetemperature of the whole broth hydrolysate can be in a range from 45° C.to 65° C., or even from 50° C. to 60° C. In some embodiments, the pH ofthe whole broth hydrolysate can be in a range from 4 to 6, or even from4.5 to 5.5. In some embodiments, the saccharification time period is inthe range from 48 to 120 hours, or even from 112 to 114 hours.

After enzymatic hydrolysis in system 135, stream 145 can be fed intofermentation system 150 so that yeast from propagation system 140 canconvert xylose and glucose into ethanol. The beer 155 from fermentationsystem 150 can be fed to distillation system 160 to recover abiochemical such as ethanol.

EXAMPLE 1

Example 1 relates to the graphs in FIGS. 3A-5C, which show thatalpha-amylase at different concentrations (1T=0.007% w/w as-is corn,2T=0.013% w/w as-is corn, 3T=0.025% w/w as-is corn, and 4T=0.05% w/was-is corn) is effective at generating glucose after cooling to 31.1° C.and adding gluco-amylase (1T=0.012% w/w as-is corn, 2T=0.025% w/w as-iscorn, 3T=0.035% w/w as-is corn, and 4T=0.05% w/w as-is corn).Pasteurization was performed at 70° C., 75° C., and 80° C. for twohours. This process was able to approach the theoretical maximum sugarrelease despite the enzymes not being at their optimal conditions. Theflat, dashed line in each of FIGS. 3A-5C represents the theoreticalmaximum sugar concentration.

EXAMPE 2

Example 2 illustrates how incremental gluco-amylase may be added to apropagation media to create the exponential sugar release profile shownin FIG. 6. The benefit of the exponential sugar release compared to thelogarithmic sugar release shown in Example 1 with alpha-amylase andgluco-amylase is limitation of sugar going to ethanol due to theCrabtree Effect. The Crabtree Effect is the production of ethanol andCO₂ during aerobic conditions due to high sugar concentrations. Theproduction of ethanol would reduce the amount of sugar going to cellmass.

EXAMPLE 3

Shown in FIGS. 7A-7C are lab scale main fermentation results using thesame fermentation medium comparing the use of propagated cells usingstarch based propagation media and propagated cells using cellulosichydrolysate based propagation media. Both propagations usedapproximately the same total sugar concentration. The results showedthat when using propagated cells using starch based propagation media,greater xylose is consumed, greater ethanol is produced, and less lacticacid (from bacterial contamination) is produced. Furthermore, bothxylose consumption and ethanol production occur at higher rates.

What is claimed is:
 1. A method of producing a biochemical, the methodcomprising: a) providing a propagation composition comprising one ormore saccharides derived from grain, and a first cell mass of amicroorganism; b) exposing the propagation composition to conditions topropagate the first cell mass of the microorganism into a second cellmass of the microorganism greater than the first cell mass of themicroorganism; c) providing a fermentation composition comprising one ormore saccharides derived from lignocellulosic feedstock and one or moretoxins that are toxic to the microorganism, and at least a portion ofthe second cell mass of microorganism; and d) exposing the fermentationcomposition to conditions so that the at least a portion of the secondcell mass of microorganism converts saccharides in the fermentationcomposition into a biochemical.
 2. The method of claim 1, wherein thepropagation composition comprises a ground grain slurry, wherein theground grain slurry comprises one or more polysaccharides.
 3. The methodof claim 2, wherein the one or more polysaccharides comprise starch, andwherein the ground grain slurry is formed from grain chosen from corn,barley, wheat, sorghum, and combinations thereof.
 4. The method of claim2, wherein the ground grain slurry comprises ground corn.
 5. The methodof claim 4, wherein the ground corn is formed by dry grinding wholecorn.
 6. The method of claim 1, wherein the saccharides in thepropagation composition comprise starch, and wherein the propagationcomposition further comprises one or more enzymes chosen fromalpha-amylase, glucoamylase, and combinations thereof.
 7. The method ofclaim 1, wherein the microorganism is chosen from bacteria, fungi, andcombinations thereof.
 8. The method of claim 1, wherein themicroorganism is from a genus chosen from Aspergillus, Candida, Pichia,(Hansenula), Phanerochaete, Kloeckera (Hanseniaspora), Kluyveromyces,Rhodotorula, Torulopsis, Zygosaccharomyces, Yarrowia, Saccharomyces, andcombinations thereof.
 9. The method of claim 1, wherein themicroorganism comprises Saccharomyces cerevisiae.
 10. The method ofclaim 1, wherein the biochemical is chosen from ethanol, butanol, andcombinations thereof.
 11. The method of claim 1, wherein the propagationcomposition further comprises a grain stillage composition, wherein thegrain stillage composition is derived from distilling a corn grainfermentation product, and wherein the grain stillage compositioncomprises whole stillage, thin stillage, and/or syrup.
 12. The method ofclaim 1, wherein the one or more toxins are chosen from furfural,hydroxymethylfurfural, acetic acid, and combinations thereof.
 13. Themethod of claim 1, wherein the lignocellulosic feedstock comprises cornstover and corn cobs, and the toxin comprises furfural.
 14. The methodof claim 1, wherein the one or more saccharides are formed by contactingground corn stover and ground corn cobs with an aqueous composition tohydrolyze hemicellulose into one or more pentoses, wherein the aqueouscomposition comprises an acid and has a pH from 1 to 3, and wherein theaqueous composition is at a temperature in the range from greater than110° C. to 170° C.
 15. The method of claim 14, further comprising, aftercontacting ground corn stover and ground corn cobs with an aqueouscomposition to hydrolyze hemicellulose into one or more pentoses,contacting ground corn stover and ground corn cobs with one or morecellulase enzymes to hydrolyze cellulose into glucose.
 16. A method ofpropagating a microorganism, the method comprising: a) providing apropagation composition comprising one or more polysaccharides, a firstcell mass of a microorganism that can convert monosaccharide into abiochemical, and one or more enzymes that can convert polysaccharideinto one or more monosaccharides; b) exposing the propagationcomposition to conditions to convert polysaccharide into one or moremonosaccharides, and propagate the first cell mass of the microorganisminto a second cell mass of the microorganism; c) providing alignocellulosic hydrolysate comprising one or more monosaccharides andone or more toxins that are toxic to the microorganism; and d) combiningthe lignocellulosic hydrolysate and at least a portion of the secondcell mass of microorganism so that the second cell mass of micororganismcan convert monosaccharide into a biochemical.
 17. The method of claim16, wherein the propagation composition comprises a ground grain slurry,wherein the ground grain slurry comprises the one or morepolysaccharides.
 18. The method of claim 16, wherein the microorganismis from a genus chosen from Aspergillus, Candida, Pichia, (Hansenula),Phanerochaete, Kloeckera (Hanseniaspora), Kluyveromyces, Rhodotorula,Torulopsis, Zygosaccharomyces, Yarrowia, Saccharomyces, and combinationsthereof.
 19. A biorefinery comprising: a) a propagation systemcomprising at least one vessel that contains a propagation composition,wherein the propagation composition comprises one or more saccharidesderived from grain and a first cell mass of a microorganism, wherein thepropagation system is configured to expose the propagation compositionto conditions to propagate the first cell mass of the microorganism intoa second cell mass of the microorganism greater than the first cell massof the microorganism; and b) a fermentation system, wherein thefermentation system is in fluid communication with the propagationsystem to receive at least a portion of the second cell mass of themicroorganism, wherein the fermentation system is also in fluidcommunication with a source of a fermentation composition comprisingsaccharides derived from lignocellulosic feedstock and one or moretoxins that are toxic to the microorganism, and wherein the fermentationsystem is configured to combine the fermentation composition and the atleast a portion of the second cell mass of the microorganism so that thesecond cell mass of the microorganism converts saccharide in thefermentation composition into a biochemical.
 20. The biorefinery ofclaim 19, further comprising a distillation system in fluidcommunication with the fermentation system, wherein the distillationsystem is configured to recover biochemical.